miR-27a Regulates Sheep Adipocyte Differentiation by Targeting CPT1B Gene

Simple Summary The content of intramuscular fat (IMF) is the main determinant of the nutritional and economic value of sheep meat. Therefore, lipid synthesis in sheep longissimus lumborum (LL) has become an important research focus. MicroRNA-27a (miR-27a) has been shown to play a crucial role in the proliferation and differentiation of adipocyte progenitor cells. In this study, we revealed that miR-27a significantly inhibited the formation of lipid droplets by targeting CPT1B to inhibit genes involved in lipid synthesis including PPAR γ, SCD, LPL, and FABP4. Here, we constructed a miR-27a-CPT1B regulatory network map, which revealed the interaction between miR-27a and CPT1B in lipid synthesis in ovine preadipocytes. Abstract MiRNAs are vital regulators and play a major role in cell differentiation, biological development, and disease occurrence. In recent years, many studies have found that miRNAs are involved in the proliferation and differentiation of adipocytes. The objective of this study was to evaluate the effect of miR-27a and its target gene CPT1B on ovine preadipocytes differentiation in Small-tailed Han sheep (Ovis aries). Down-regulation of miR-27a significantly promoted the production of lipid droplets, while overexpression of miR-27a led to a reduction in lipid droplet production. In addition, inhibition of miR-27a led to a significant increase in the expression of genes involved in lipid synthesis, including PPAR γ, SCD, LPL, and FABP4. Target Scan software predicted that CPT1B is a new potential target gene of miR-27a. Further experiments revealed that CPT1B gene expression and protein levels were negatively correlated with miR-27a expression. Overexpression of miR-27a led to a significant decrease in CPT1B mRNA levels and inhibited the accumulation of lipid droplets and vice versa. Moreover, overexpression of CPT1B promoted the synthesis of lipid droplets in ovine preadipocytes. Furthermore, luciferase reporter assays confirmed CPT1B to be a miR-27a direct target gene. This study confirmed that miR-27a increases the expression of genes related to lipid synthesis in ovine preadipocytes by targeting CPT1B, thereby promoting the synthesis of lipid droplets. The results of this study can be used to be exploited in devising novel approaches for improving the IMF content of sheep.


Introduction
The content of intramuscular fat (IMF) content is an index to measure meat quality [1], which directly affects the tenderness, juiciness, and flavor of mutton [2]. Therefore, understanding the mechanism of IMF synthesis in sheep meat has become a research hotspot. The formation of IMF in sheep is regulated by many factors, such as heredity [3], age [4], environment [5], and nutrition [6]. However, the molecular mechanism underlying IMF formation is still poorly understood.
For measuring intracellular lipid content, stained ovine preadipocytes were eluted with pure isopropanol for 1 h, and sample absorbance was measured at 510 nm in an Epoch spectrophotometer (BioTek, Winooski, VT, USA).

Data Analysis
Data analysis was performed in GraphPad v.8 (GraphPad Software Inc., San Diego, CA, USA). Data are presented as mean ± SD. Each experiment was performed in triplicate. Student's t-test was used for pairwise comparisons. Statistical significance was considered at values of p < 0.05, p < 0.01, and p < 0.001.

Expression of miR-27a and CPT1B under Ovine Preadipocytes Differentiation
Ovine preadipocytes were successfully isolated from the LL of Small-tailed Han sheep and submitted to induced differentiation. Temporal patterns of miR-27a and CPT1B expression were assessed during ovine preadipocytes adipogenesis. During ovine preadipocytes differentiation, expression of miR-27a gradually decreased ( Figure 1A), whereas expression of CPT1B gradually increased ( Figure 1B). These results suggest that miR-27a displayed a contrary expression pattern with CPT1B during ovine preadipocytes differentiation.

Data Analysis
Data analysis was performed in GraphPad v.8 (GraphPad Software Inc., San Diego, CA, USA). Data are presented as mean ± SD. Each experiment was performed in triplicate. Student's t-test was used for pairwise comparisons. Statistical significance was considered at values of p < 0.05, p < 0.01, and p < 0.001.

Expression of miR-27a and CPT1B under Ovine Preadipocytes Differentiation
Ovine preadipocytes were successfully isolated from the LL of Small-tailed Han sheep and submitted to induced differentiation. Temporal patterns of miR-27a and CPT1B expression were assessed during ovine preadipocytes adipogenesis. During ovine preadipocytes differentiation, expression of miR-27a gradually decreased ( Figure 1A), whereas expression of CPT1B gradually increased ( Figure 1B). These results suggest that miR-27a displayed a contrary expression pattern with CPT1B during ovine preadipocytes differentiation.

MiR-27a Inhibits Differentiation of Ovine Preadipocytes
The efficiency of miR-27a mimic and inhibitor transfection was confirmed by RT-PCR. miR-27a simulant significantly increased expression of miR-27a (p < 0.001) (Figure 2A), while miR-27a inhibitor significantly inhibited expression of miR-27a (p < 0.05) ( Figure 3A). To determine the role of miR-27a in lipid metabolism, the absorbance of the sample at 510nm was determined to measure the content of intracellular lipids, and lipid droplet accumulation following overexpression or silencing of miR-27a was determined. miR-27a mimic significantly inhibited the accumulation of lipid droplets compared with the control group (p < 0.001) ( Figure 2B,C). In contrast, the number of lipid droplets increased significantly when miR-27a was inhibited (p < 0.05) ( Figure 3B,C). Overall, these findings indicate that miR-27a inhibits the synthesis of lipid droplets in ovine preadipocytes. To determine the role of miR-27a in lipid metabolism, the absorbance of the sample at 510nm was determined to measure the content of intracellular lipids, and lipid droplet accumulation following overexpression or silencing of miR-27a was determined. miR-27a mimic significantly inhibited the accumulation of lipid droplets compared with the control group (p < 0.001) ( Figure 2B,C). In contrast, the number of lipid droplets increased significantly when miR-27a was inhibited (p < 0.05) ( Figure 3B,C). Overall, these findings indicate that miR-27a inhibits the synthesis of lipid droplets in ovine preadipocytes.

CPT1B Is a Target Gene for miR-27a
As shown in Figure 5A, treatment with miR-27a mimic led to downregulation of CPT1B expression. In contrast, treatment with miR-27a inhibitor remarkably enhanced CPT1B expression (p < 0.01) ( Figure 5B). Simultaneously, Western blot assay revealed that levels of CPT1B protein in miR-27a mimic and inhibitor transfection groups were comparable to those of mRNA expression. Therefore, it can be stated that miR-27a reduced CPT1B mRNA levels and protein levels in ovine preadipocytes.
To further investigate the mechanism underlying miR-27a inhibition of fat deposition in sheep muscle tissue, we aimed to investigate whether miR-27a targets the 3'-UTR region of CPT1B, this region had the binding site for the seed sequence of miR-27a (Figure

CPT1B Is a Target Gene for miR-27a
As shown in Figure 5A, treatment with miR-27a mimic led to downregulation of CPT1B expression. In contrast, treatment with miR-27a inhibitor remarkably enhanced CPT1B expression (p < 0.01) ( Figure 5B). Simultaneously, Western blot assay revealed that levels of CPT1B protein in miR-27a mimic and inhibitor transfection groups were comparable to those of mRNA expression. Therefore, it can be stated that miR-27a reduced CPT1B mRNA levels and protein levels in ovine preadipocytes. target gene for miR-27a. Co-transfection of miR-27a mimics and CPT1B-WT-miR-27a vector directly repressed luciferase activity ( Figure 6B; p < 0.01), which suggests that CPT1B is a direct target gene of miR-27a. Overall, these findings revealed that miR-27a inhibited differentiation and promoted proliferation in ovine preadipocytes by negatively regulating CPT1B. To further investigate the mechanism underlying miR-27a inhibition of fat deposition in sheep muscle tissue, we aimed to investigate whether miR-27a targets the 3'-UTR region of CPT1B, this region had the binding site for the seed sequence of miR-27a ( Figure 6A). Therefore, the luciferase reporter system was used to confirm whether CPT1B is a target gene for miR-27a. Co-transfection of miR-27a mimics and CPT1B-WT-miR-27a vector directly repressed luciferase activity ( Figure 6B; p < 0.01), which suggests that CPT1B is a direct target gene of miR-27a. Overall, these findings revealed that miR-27a inhibited differentiation and promoted proliferation in ovine preadipocytes by negatively regulating CPT1B.

Effect of CPT1B Overexpression on Ovine Preadipocytes Differentiation
To reveal the role of CPT1B in lipid metabolism of ovine preadipocytes, a CPT1B overexpression vector was constructed and transfected into ovine preadipocytes. The optimal transfection concentration was 2.5 µg to yield the highest expression efficiency. Compared with the control group, levels of CPT1B mRNA and protein expression in transfected ovine preadipocytes increased significantly (p < 0.05) ( Figure 7A,B). After 8 days of induction, CPT1B overexpression promoted lipid accumulation in ovine preadipocytes ( Figure 7C). Collectively, these findings suggest that overexpression of CPT1B in ovine preadipocytes promotes adipogenesis.

Effect of CPT1B Overexpression on Ovine Preadipocytes Differentiation
To reveal the role of CPT1B in lipid metabolism of ovine preadipocytes, a CPT1B overexpression vector was constructed and transfected into ovine preadipocytes. The optimal transfection concentration was 2.5 µg to yield the highest expression efficiency. Compared with the control group, levels of CPT1B mRNA and protein expression in transfected ovine preadipocytes increased significantly (p < 0.05) ( Figure 7A,B). After 8 days of induction, CPT1B overexpression promoted lipid accumulation in ovine preadipocytes ( Figure 7C). Collectively, these findings suggest that overexpression of CPT1B in ovine preadipocytes promotes adipogenesis.

Discussion
CPT1B is a major isotype of the CPT family which comprises other two genes, namely CPT1A and CPT2 [23]. CPT1B gene plays a vital role in regulating fat decomposition and energy supply. Studies have shown that CPT1B is of great significance to the development of brown adipose tissue [24]. Moreover, in bovine fetal fibroblasts, changes in triglyceride content were related to the expression level of CPT1B [25]. It has also been demonstrated that downregulation of CPT1B leads to impaired fatty acid oxidation [26]. Furthermore, decreased CPT1B protein levels limit the oxidation of cardiac fatty acids [27]. In the present study, overexpression of CPT1B led to a significant increase in lipid accumulation in ovine preadipocytes. Interestingly, previous studies have reported that miR-27a inhibits ovine preadipocytes differentiation by regulating the expression of the target gene RXR alpha [13]. CPT1B is downstream RXR alpha in the PPAR γ signaling pathway. Therefore, it can be speculated that miR-27a may regulate lipid deposition in ovine preadipocytes via the PPAR γ signaling pathway. However, the specific mechanism of regulation needs further elucidation. Based on the above-described results, it can be inferred that miR-27a regulates lipid deposition in ovine preadipocytes by targeting CPT1B. Animal fat metabolism is regulated by many factors [28][29][30]. Abnormal fat metabolism leads to increased incidence of a variety of metabolic diseases [31,32]. Therefore, understanding the mechanism underlying the regulation of lipid metabolism has always been the focus of several studies. In recent years, research on miRNA has confirmed that a large number of miRNAs are involved in the regulation of many biological processes related to adipose tissue development [33][34][35]. In previous studies, it has been suggested that CPT1B can be a candidate gene for fat deposition in sheep adipose tissue. In addition, it has been hypothesized previously that CPT1B may be a potential target gene for miR-27a. In the present study, ovine preadipocytes were employed as a model to explore the regulatory mechanism between miR-27a and CPT1B in adipose tissue differentiation, which provided a theoretical basis for further understanding the effect of miRNA on lipid formation in ovine preadipocytes.
The regulatory effect of miR-27a on many types of cells has been previously reported. For instance, silencing miR-27a promoted autophagy and apoptosis of melanoma cells [36], whereas overexpression of miR-27a targeted GSK-3 β and promoted breast epithelial cell proliferation and invasion [37]. Moreover, miR-27a-3p reduced ATF3 mRNA and protein levels in a cellular vascular calcification model. [38]. Additionally, miR-27a is closely related to lipid synthesis [39]. MiR-27a affects lipid deposition in IMF and subcutaneous adipose tissue of sheep by participating in adipocyte differentiation and triglyceride synthesis [40,41]. miR-27a and miR-27b target expression of PPAR γ in 3T3-L1 cells, thereby controlling the production of lipid droplets [42]. MiR27a can also inhibit NRF2 expression and contribute to lipid accumulation in hepatocytes [43]. Collectively, the results discussed herein contribute to show that miR-27a negatively regulates lipid production in ovine preadipocytes.
In addition, in the present study, the expression level of miR-27a decreased with differentiation and maturation of ovine preadipocytes, whereas the expression level of CPT1B increased with the differentiation and maturation of ovine preadipocytes. These findings are consistent with previous works which showed that the expression level of the CPT1B gene increased with an increase in the accumulation of lipid droplets in bovine fetal fibroblasts [25]. The expression trend of miR-27a is opposite to the marker gene PPAR γ for adipogenic differentiation [44,45], whereas PPAR γ expression level is positively correlated with ovine preadipocytes differentiation. Collectively, these results suggest that miR-27a negatively regulates differentiation of and lipid accumulation in ovine preadipocytes.
Herein, miR-27a was found to inhibit significantly lipid droplet accumulation in ovine preadipocytes, which is consistent with previous studies showing that miR-27a negatively regulates lipid production in ovine preadipocytes [13]. Furthermore, miR-27a was shown to decrease expression of PPAR γ, SCD, LPL, and FABP4 which are genes related to lipid synthesis in ovine preadipocytes. PPAR γ is a positive regulator of lipid accumulation which plays a key regulatory role in adipocyte differentiation [46]. FABP4 has a significant role in fat deposition and is highly expressed in fat tail [47]. Previous studies showed a possible effect of SCD on the regulation of milk fat percentage in Churra sheep [48]. LPL plays an important role in regulating fat synthesis in goat breast milk [49]. Our results, therefore, suggest that miR-27a may inhibit the synthesis of lipid droplets by affecting key genes involved in lipid regulation.

Conclusions
In the present study, miR-27a was shown to be involved in fat synthesis and accumulation in ovine preadipocytes. Specifically, miR-27a regulated the formation of lipid droplets in adipocytes by targeting the CPT1B gene. These results may provide a theoretical basis for understanding IMF deposition in sheep muscle tissue.

Institutional Review Board Statement:
The study was conducted according to the institutional animal care and committee (IACUC) guidelines and approved by the Animal Care and Use Committee of Northwest A&F University, Yangling, China (NWAFU-20201212).

Data Availability Statement:
The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.